Phorbol myristate acetate inhibits thrombin-stimulated Ca2+ mobilization and phosphatidylinositol 4,5-bisphosphate hydrolysis in human platelets.

Abstract

The tumor-promoting phorbol diester 4.beta.-phorbol 12-myristate 13-acetate (PMA) inhibited mobilization of intracellular Ca2+ in [human] platelets by thrombin (also trypsin and 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphocholine). PMA was effective over the same concentration range that activates protein kinase C in intact platelets; IC50 vs. thrombin = 2 ng/ml, 3.4 nM: > 90% inhibition at 10-20 ng/ml. Suppression of thrombin-induced Ca2+ mobilization was evident within 30 s of pretreatment with PMA and was essentially complete by 6-10 min at 10-20 ng PMA/ml. Thrombin-induced secretion was initially accelerated in the presence of PMA, but after 1 min it was progressively inhibited when Ca2+ mobilization was depressed by > 60%. PMA did not inhibit Ca2+ mobilization or secretion caused by A23187. Thrombin-induced phosphatidylinositol 4,5-[32P]bisphosphate breakdown and [32P]phosphatidic acid production were also initially increased by PMA and then progressively depressed. Inhibition of thrombin-induced lipid metabolism required higher concentrations of PMA (IC50 = 10 ng/ml), and it was not overcome by A23187. 4.alpha.-Phorbol 12,13-didecanoate, which lacks the ability to activate protein kinase C, did not inhibit any responses to thrombin. Activation of protein kinase C, which initially fosters secretion and aggregation, may subsequently exert negative feedback on the receptor-mediated mobilization of intracellular Ca2+ and the hydrolysis of phosphatidylinositol 4,5-biphosphate.